Gene coding for glucose-6-phosphate-dehydrogenase proteins

ABSTRACT

The present invention relates to isolated nucleic acid molecules encoding mutants of glucose-6-phosphate dehydrogenase, and vectors and hosts cells including such nucleic acid molecules. These nucleic acid molecules are involved in the biosynthesis of a fine chemical, e.g., an amino acid such as lysine. The present invention also relates to methods of producing and modulating the production of fine chemicals, e.g., lysine, by culturing recombinant microorganisms containing these nucleic acid molecules under conditions such that the fine chemical is produced.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/055,939, filed Mar. 26, 2008, which is a continuation of U.S.application Ser. No. 11/787,137, filed Apr. 13, 2007, which is acontinuation of U.S. application Ser. No. 10/495,291, filed May 11,2004, which is a 35 U.S.C. 371 National stage filing of InternationalApplication No. PCT/EP02/12556, filed Nov. 11, 2002, which claimspriority to German Application No. 101 55 505.9, filed Nov. 13, 2001.The entire contents of each of these applications are herebyincorporated by reference herein.

SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_List_(—)12810_(—)01049_US. The size ofthe text file is 23 KB, and the text file was created on Jul. 23, 2010.

BACKGROUND OF THE INVENTION

Particular products and byproducts of naturally occurring metabolicprocesses in cells are used in many branches of industry, including thefood industry, the animal feed industry, the cosmetics industry and thepharmaceutical industry. These molecules which are collectively referredto as “fine chemicals” comprise organic acids, both proteinogenic andnonproteinogenic amino acids, nucleotides and nucleosides, lipids andfatty acids, diols, carbohydrates, aromatic compounds, vitamins,cofactors and enzymes. They are best produced by means of cultivating,on a large scale, bacteria which have been developed to produce andsecrete large amounts of the molecule desired in each particular case.An organism which is particularly suitable for this purpose isCorynebacterium glutamicum, a Gram-positive nonpathogenic bacterium.Using strain selection, a number of mutant strains have been developedwhich produce various desirable compounds. The selection of strainswhich are improved with respect to the production of a particularmolecule is, however, a time-consuming and difficult process.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides novel nucleic acid molecules which can beused for identifying or classifying Corynebacterium glutamicum orrelated bacterial species. C. glutamicum is a Gram-positive, aerobicbacterium which is widely used in industry for the large-scaleproduction of a number of fine chemicals and also for the degradation ofhydrocarbons (for example in the case of crude oil spills) and for theoxidation of terpenoids. The nucleic acid molecules may therefore beused for identifying microorganisms which can be used for producing finechemicals, for example by fermentation processes. Although C. glutamicumitself is nonpathogenic, it is, however, related to otherCorynebacterium species such as Corynebacterium diphteriae (thediphtheria pathogen), which are major pathogens in humans. The abilityto identify the presence of Corynebacterium species may therefore alsobe of significant clinical importance, for example in diagnosticapplications. Moreover, said nucleic acid molecules may serve asreference points for mapping the C. glutamicum genome or genomes ofrelated organisms.

These novel nucleic acid molecules encode proteins which are referred toas glucose-6-phosphate-dehydrogenase proteins.

Glucose-6-phosphate-dehydrogenase genes from Corynebacteria aredescribed, for example, in EP 1108790A2. However, the genes describedtherein code for polypeptide sequences which are shorter than thosedescribed herein according to the invention. The N terminus of theglucose-6-phosphate-dehydrogenase described in EP 1108790A2 is truncatedby 30 amino acids compared with the polypeptide sequence claimed herein.

Moritz et al. (Eur. J. Biochemistry 267, 3442-3452, 2000) describe theisolation of a glucose-6-phosphate-dehydrogenase from Corynebacteriumglutamicum. The N-terminal protein sequencing described therein resultsin a polypeptide which starts with a serine and differs from the proteinof the invention.

The invention relates to novel genes forglucose-6-phosphate-dehydrogenase, which start with the amino acid atposition 1 or 2, i.e. Val or Ser and which encode at position 243 aproteinogenic amino acid which is not Ala (numbering based on SEQ ID NO:2).

Particular preference is given to novel genes forglucose-6-phosphate-dehydrogenase, which start with the amino acid atposition 1 and encode Thr at position 243 (numbering based on SEQ ID NO:2).

The nucleic acid molecules of the invention can be used for geneticmanipulation of an organism in order to make it a better and moreefficient producer of one or more fine chemicals. The molecules of theinvention can be modified so as to improve the yield, production and/orefficiency of production of one or more fine chemicals.

Furthermore, the molecules of the invention may be involved in one ormore intracellular signal transduction pathways which influence theyields and/or the rate of production of one or more fine chemicals fromC. glutamicum. Proteins which are required, for example, for importingone or more sugars from the extracellular medium (e.g. Hpr, enzyme I ora component of the enzyme II complex) are, if a sufficient amount ofsugar is present in the cell, frequently posttranslationally modified sothat they are no longer able to import said sugar. Although the amountof sugar at which the transport system is switched off is sufficient formaintaining normal cellular functions, it limits overproduction of thefine chemical of interest. It is therefore recommended to modify theproteins of the invention so that they no longer respond to such anegative regulation. As a result, it is possible to attain higherintracellular concentrations of one or more sugars and, by extension, amore efficient production or higher yields of one or more fine chemicalsfrom organisms which contain said mutant proteins.

Appendix A defines hereinbelow the nucleic acid sequences of thesequence listing together with the sequence modifications at therelevant position, described in Table 1.

Appendix B defines hereinbelow the polypeptide sequences of the sequencelisting together with the sequence modifications at the relevantposition, described in Table 1.

In a further embodiment, the isolated nucleic acid molecule is at least15 nucleotides in length and hybridizes under stringent conditions to anucleic acid molecule which comprises a nucleotide sequence of AppendixA. The isolated nucleic acid molecule preferably corresponds to anaturally occurring nucleic acid molecule. The isolated nucleic acidmore preferably encodes a naturally occurring C. glutamicum G6PD proteinor a biologically active section thereof.

A further aspect of the invention relates to vectors, for examplerecombinant expression vectors, which contain the nucleic acid moleculesof the invention and to host cells into which said vectors have beenintroduced. In one embodiment, a G6PD protein is prepared by using ahost cell which is cultivated in a suitable medium. The G6PD protein maythen be isolated from the medium or the host cell.

A further aspect of the invention relates to a genetically modifiedmicroorganism into which a G6PD gene has been introduced or in which aG6PD gene has been modified. In one embodiment, the genome of saidmicroorganism has been modified by introducing at least one inventivenucleic acid molecule which encodes the mutated G6PD sequence astransgene. In another embodiment, an endogenous G6PD gene in the genomeof said microorganism has been modified, for example, functionallydisrupted, by homologous recombination with a modified G6PD gene. In apreferred embodiment, the microorganism belongs to the genusCorynebacterium or Brevibacterium, with Corynebacterium glutamicum beingparticularly preferred. In a preferred embodiment, the microorganism isalso used for preparing a compound of interest, such as an amino acid,particularly preferably lysine.

Another preferred embodiment are host cells having more than one of thenucleic acid molecules described in Appendix A. Such host cells can beprepared in various ways known to the skilled worker. They may betransfected, for example, by vectors carrying several of the nucleicacid molecules of the invention. However, it is also possible to use avector for introducing in each case one nucleic acid molecule of theinvention into the host cell and therefore to use a plurality of vectorseither simultaneously or sequentially. Thus it is possible to constructhost cells which carry numerous, up to several hundred, nucleic acidsequences of the invention. Such an accumulation can often producesuperadditive effects on the host cell with respect to fine-chemicalproductivity.

A further aspect of the invention relates to an isolated G6PD protein ora section thereof, for example a biologically active section. In apreferred embodiment, the isolated G6PD protein or its section may beinvolved in importing energy-rich carbon molecules (e.g. glucose,fructose or sucrose) into C. glutamicum and, moreover, in one or moreintracellular signal transduction pathways of C. glutamicum. In anotherpreferred embodiment, the isolated G6PD protein or a section thereof issufficiently homologous to an amino acid sequence of Appendix B so thatthe protein or its section is still capable of taking part in importingenergy-rich carbon molecules (e.g. glucose, fructose or sucrose) into C.glutamicum and/or in one or more intracellular signal transductionpathways of C. glutamicum.

Moreover, the invention relates to an isolatedglucose-6-phosphate-dehydrogenase protein preparation. In preferredembodiments, the glucose-6-phosphate-dehydrogenase (G6PD) proteincomprises an amino acid sequence of Appendix B. In a further preferredembodiment, the invention relates to an isolated full-length proteinwhich is essentially homologous to a complete amino acid sequence ofAppendix B (which is encoded by an open reading frame in Appendix A).

A further aspect of the invention relates to a method for preparing afine chemical. The method provides for the cultivation of a cellcontaining a vector which causes expression of a nucleic acid moleculeof the invention so that a fine chemical is produced. In a preferredembodiment, this method moreover comprises the step of obtaining a cellcontaining such a vector, said cell being transfected with a vectorwhich causes expression of a nucleic acid. In a further preferredembodiment, said method moreover comprises the step in which the finechemical is obtained from the culture. In a preferred embodiment, thecell belongs to the genus Corynebacterium or Brevibacterium.

I. Fine Chemicals

The term “fine chemicals” is known in the art and includes moleculeswhich are produced by an organism and are used in various branches ofindustry such as, for example, but not restricted to, the pharmaceuticalindustry, the agricultural industry and the cosmetics industry. Thesecompounds comprise organic acids such as tartaric acid, itaconic acidand diaminopimelic acid, both proteinogenic and nonproteinogenic aminoacids, purine and pyrimidine bases, nucleosides and nucleotides (asdescribed, for example, in Kuninaka, A. (1996) Nucleotides and relatedcompounds, pp. 561-612, in Biotechnology Vol. 6, Rehm et al., EditorsVCH: Weinheim and the references therein), lipids, saturated andunsaturated fatty acids (e.g. arachidonic acid), diols (e.g. propanedioland butanediol), carbohydrates (e.g. hyaluronic acid and trehalose),aromatic compounds (e.g. aromatic amines, vanilline and indigo),vitamins and cofactors (as described in Ullmann's Encyclopedia ofIndustrial Chemistry, Vol. A27, “Vitamins”, pp. 443-613 (1996) VCH:Weinheim and the references therein; and Ong, A. S., Niki, E. andPacker, L. (1995) “Nutrition, Lipids, Health and Disease” Proceedings ofthe UNESCO/Confederation of Scientific and Technological Associations inMalaysia and the Society for Free Radical Research—Asia, held Sep. 1-3,1994 in Penang, Malaysia, AOCS Press (1995)), enzymes and all otherchemicals described by Gutcho (1983) in Chemicals by Fermentation, NoyesData Corporation, ISBN: 0818805086 and the references indicated therein.The metabolism and the uses of particular fine chemicals are furtherillustrated below.

A. Amino Acid Metabolism and Uses

Amino acids comprise the fundamental structural units of all proteinsand are thus essential for normal functions of the cell. The term “aminoacid” is known in the art. Proteinogenic amino acids, of which there are20 types, serve as structural units for proteins, in which they arelinked together by peptide bonds, whereas the nonproteinogenic aminoacids (hundreds of which are known) usually do not occur in proteins(see Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2, pp. 57-97VCH: Weinheim (1985)). Amino acids can exist in the D or Lconfiguration, although L-amino acids are usually the only type found innaturally occurring proteins. Biosynthetic and degradation pathways ofeach of the 20 proteinogenic amino acids are well characterized both inprokaryotic and eukaryotic cells (see, for example, Stryer, L.Biochemistry, 3^(rd) edition, pp. 578-590 (1988)). The “essential” aminoacids (histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan and valine), so called because,owing to the complexity of their biosyntheses, they must be taken inwith the diet, are converted by simple biosynthetic pathways into theother 11 “nonessential” amino acids (alanine, arginine, asparagine,aspartate, cysteine, glutamate, glutamine, glycine, proline, serine andtyrosine). Higher animals are able to synthesize some of these aminoacids but the essential amino acids must be taken in with the food inorder that normal protein synthesis takes place.

Apart from their function in protein biosynthesis, these amino acids areinteresting chemicals as such, and it has been found that many havevarious applications in the human food, animal feed, chemicals,cosmetics, agricultural and pharmaceutical industries. Lysine is animportant amino acid not only for human nutrition but also formonogastric livestock such as poultry and pigs. Glutamate is mostfrequently used as flavor additive (monosodium glutamate, MSG) andelsewhere in the food industry, as are aspartate, phenylalanine, glycineand cysteine. Glycine, L-methionine and tryptophan are all used in thepharmaceutical industry. Glutamine, valine, leucine, isoleucine,histidine, arginine, proline, serine and alanine are used in thepharmaceutical industry and the cosmetics industry. Threonine,tryptophan and D/L-methionine are widely used animal feed additives(Leuchtenberger, W. (1996) Amino acids—technical production and use, pp.466-502 in Rehm et al., (editors) Biotechnology Vol. 6, Chapter 14a,VCH: Weinheim). It has been found that these amino acids areadditionally suitable as precursors for synthesizing synthetic aminoacids and proteins, such as N-acetylcysteine,S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan and other substancesdescribed in Ullmann's Encyclopedia of Industrial Chemistry, Vol. A2,pp. 57-97, VCH, Weinheim, 1985.

The biosynthesis of these natural amino acids in organisms able toproduce them, for example bacteria, has been well characterized (for areview of bacterial amino acid biosynthesis and its regulation, seeUmbarger, H. E. (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate issynthesized by reductive amination of □-ketoglutarate, an intermediateproduct in the citric acid cycle. Glutamine, proline and arginine areeach generated successively from glutamate. The biosynthesis of serinetakes place in a three-step process and starts with 3-phosphoglycerate(an intermediate product of glycolysis), and affords this amino acidafter oxidation, transamination and hydrolysis steps. Cysteine andglycine are each produced from serine, specifically the former bycondensation of homocysteine with serine, and the latter by transfer ofthe side-chain □-carbon atom to tetrahydrofolate in a reaction catalyzedby serine transhydroxy-methylase. Phenylalanine and tyrosine aresynthesized from the precursors of the glycolysis and pentose phosphatepathway, and erythrose 4-phosphate and phosphoenolpyruvate in a 9-stepbiosynthetic pathway which diverges only in the last two steps after thesynthesis of prephenate. Tryptophan is likewise produced from these twostarting molecules but it is synthesized by an 11-step pathway. Tyrosinecan also be prepared from phenylalanine in a reaction catalyzed byphenylalanine hydroxylase. Alanine, valine and leucine are eachbiosynthetic products derived from pyruvate, the final product ofglycolysis. Aspartate is formed from oxalacetate, an intermediateproduct of the citrate cycle. Asparagine, methionine, threonine andlysine are each produced by the conversion of aspartate. Isoleucine isformed from threonine. Histidine is formed from 5-phosphoribosyl1-pyrophosphate, an activated sugar, in a complex 9-step pathway.

Amounts of amino acids exceeding those required for protein biosynthesisby the cell cannot be stored and are instead broken down so thatintermediate products are provided for the principal metabolic pathwaysin the cell (for a review, see Stryer, L., Biochemistry, 3^(rd) edition,Chapter 21 “Amino Acid Degradation and the Urea Cycle”; pp. 495-516(1988)). Although the cell is able to convert unwanted amino acids intothe useful intermediate products of metabolism, production of aminoacids is costly in terms of energy, the precursor molecules and theenzymes necessary for their synthesis. It is therefore not surprisingthat amino acid biosynthesis is regulated by feedback inhibition,whereby the presence of a particular amino acid slows down or completelystops its own production (for a review of the feedback mechanism inamino acid biosynthetic pathways, see Stryer, L., Biochemistry, 3^(rd)edition, Chapter 24, “Biosynthesis of Amino Acids and Heme”, pp. 575-600(1988)). The output of a particular amino acid is therefore restrictedby the amount of this amino acid in the cell.

B. Vitamins, Cofactors and Nutraceutical Metabolism, and Uses

Vitamins, cofactors and nutraceuticals comprise another group ofmolecules. Higher animals have lost the ability to synthesize them andtherefore have to take them in, although they are easily synthesized byother organisms such as bacteria. These molecules are either bioactivemolecules per se or precursors of bioactive substances which serve aselectron carriers or intermediate products in a number of metabolicpathways. Besides their nutritional value, these compounds also have asignificant industrial value as colorants, antioxidants and catalysts orother processing auxiliaries. (For a review of the structure, activityand industrial applications of these compounds, see, for example,Ullmann's Encyclopedia of Industrial Chemistry, “Vitamins”, Vol. A27,pp. 443-613, VCH: Weinheim, 1996). The term “vitamin” is known in theart and comprises nutrients which are required for normal functional ofan organism but cannot be synthesized by this organism itself. The groupof vitamins may include cofactors and nutraceutical compounds. The term“cofactor” comprises nonproteinaceous compounds necessary for theappearance of a normal enzymic activity. These compounds may be organicor inorganic; the cofactor molecules of the invention are preferablyorganic. The term “nutraceutical” comprises food additives which arehealth-promoting in plants and animals, especially humans. Examples ofsuch molecules are vitamins, antioxidants and likewise certain lipids(e.g. polyunsaturated fatty acids).

The biosynthesis of these molecules in organisms able to produce them,such as bacteria, has been comprehensively characterized (Ullmann'sEncyclopedia of Industrial Chemistry, “Vitamins”, Vol. A27, pp. 443-613,VCH: Weinheim, 1996, Michal, G. (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley & Sons; Ong, A. S., Niki,E. and Packer, L. (1995) “Nutrition, Lipids, Health and Disease”Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia and the Society for free Radical Research—Asia,held on Sep. 1-3, 1994, in Penang, Malaysia, AOCS Press, Champaign, Ill.X, 374 S).

Thiamine (vitamin B₁) is formed by chemical coupling of pyrimidine andthiazole units. Riboflavin (vitamin B₂) is synthesized from guanosine5′-triphosphate (GTP) and ribose 5′-phosphate. Riboflavin in turn isemployed for the synthesis of flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD). The family of compounds together referred toas “vitamin B6” (for example pyridoxine, pyridoxamine, pyridoxal5′-phosphate and the commercially used pyridoxine hydrochloride), areall derivatives of the common structural unit5-hydroxy-6-methylpyridine. Panthothenate (pantothenic acid,R-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-□-alanine) can beprepared either by chemical synthesis or by fermentation. The last stepsin pantothenate biosynthesis consist of ATP-driven condensation of□-alanine and pantoic acid. The enzymes responsible for the biosyntheticsteps for the conversion into pantoic acid and into □-alanine and forthe condensation to pantothenic acid are known. The metabolically activeform of pantothenate is coenzyme A whose biosynthesis takes place by 5enzymatic steps. Pantothenate, pyridoxal 5′-phosphate, cysteine and ATPare the precursors of coenzyme A. These enzymes catalyze not only theformation of pantothenate but also the production of (R)-pantoic acid,(R)-pantolactone, (R)-panthenol (provitamin B₅), pantetheine (and itsderivatives) and coenzyme A.

The biosynthesis of biotin from the precursor molecule pimeloyl-CoA inmicroorganisms has been investigated in detail, and several of the genesinvolved have been identified. It has emerged that many of thecorresponding proteins are involved in the Fe cluster synthesis andbelong to the class of nifS proteins. Liponic acid is derived fromoctanoic acid and serves as coenzyme in energy metabolism where it is aconstituent of the pyruvate dehydrogenase complex and of the□-ketoglutarate dehydrogenase complex. Folates are a group of substancesall derived from folic acid which in turn is derived from L-glutamicacid, p-aminobenzoic acid and 6-methylpterin. The biosynthesis of folicacid and its derivatives starting from the metabolic intermediateproducts of the biotransformation of guanosine 5′-triphosphate (GTP),L-glutamic acid and p-aminobenzoic acid has been investigated in detailin certain microorganisms.

Corrinoids (such as the cobalamines and, in particular, vitamin B₁₂) andthe porphyrins belong to a group of chemicals distinguished by atetrapyrrole ring system. The biosynthesis of vitamin B₁₂ is so complexthat it has not yet been completely characterized, but many of theenzymes and substrates involved are now known. Nicotinic acid(nicotinate) and nicotinamide are pyridine derivatives which are alsoreferred to as “niacin”. Niacin is the precursor of the importantcoenzymes NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamideadenine dinucleotide phosphate) and their reduced forms.

Production of these compounds on the industrial scale is mostly based oncell-free chemical syntheses, although some of these chemicals havelikewise been produced by large-scale cultivation of microorganisms,such as riboflavin, vitamin B₆, pantothenate and biotin. Only vitaminB₁₂ is, because of the complexity of its synthesis, produced only byfermentation. In vitro processes require a considerable expenditure ofmaterials and time and frequently high costs.

C. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses

Genes for purine and pyrimidine metabolism and their correspondingproteins are important aims for the therapy of oncoses and viralinfections. The term “purine” or “pyrimidine” comprisesnitrogen-containing bases which form part of nucleic acids, coenzymesand nucleotides. The term “nucleotide” encompasses the fundamentalstructural units of nucleic acid molecules, which comprise anitrogen-containing base, a pentose sugar (the sugar is ribose in thecase of RNA and the sugar is D-deoxyribose in the case of DNA) andphosphoric acid. The term “nucleoside” comprises molecules which serveas precursors of nucleotides but have, in contrast to the nucleotides,no phosphoric acid unit. It is possible to inhibit RNA and DNA synthesisby inhibiting the biosynthesis of these molecules or their mobilizationto form nucleic acid molecules; targeted inhibition of this activity incancerous cells allows the ability of tumor cells to divide andreplicate to be inhibited.

There are also nucleotides which do not form nucleic acid molecules butserve as energy stores (i.e. AMP) or as coenzymes (i.e. FAD and NAD).

Several publications have described the use of these chemicals for thesemedical indications, the purine and/or pyrimidine metabolism beinginfluenced (for example Christopherson, R. I. and Lyons, S. D. (1990)“Potent inhibitors of de novo pyrimidine and purine biosynthesis aschemotherapeutic agents”, Med. Res. Reviews 10: 505-548). Investigationsof enzymes involved in purine and pyrimidine metabolism haveconcentrated on the development of novel medicaments which can be used,for example, as immunosuppressants or antiproliferative agents (Smith,J. L. “Enzymes in Nucleotide Synthesis” Curr. Opin. Struct. Biol. 5(1995) 752-757; Simmonds, H. A., Biochem. Soc. Transact. 23 (1995)877-902). However, purine and pyrimidine bases, nucleosides andnucleotides also have other possible uses: as intermediate products inthe biosynthesis of various fine chemicals (e.g. thiamine,S-adenosylmethionine, folates or riboflavin), as energy carriers for thecell (for example ATP or GTP) and for chemicals themselves, areordinarily used as flavor enhancers (for example IMP or GMP) or for manymedical applications (see, for example, Kuninaka, A., (1996)“Nucleotides and Related Compounds in Biotechnology” Vol. 6, Rehm etal., editors VCH: Weinheim, pp. 561-612). Enzymes involved in purine,pyrimidine, nucleoside or nucleotide metabolism are also increasinglyserving as targets against which chemicals are being developed for cropprotection, including fungicides, herbicides and insecticides.

The metabolism of these compounds in bacteria has been characterized(for reviews, see, for example, Zalkin, H. and Dixon, J. E. (1992) “Denovo purine nucleotide biosynthesis” in Progress in Nucleic AcidsResearch and Molecular biology, Vol. 42, Academic Press, pp. 259-287;and Michal, G. (1999) “Nucleotides and Nucleosides”; Chapter 8 in:Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology,Wiley, N.Y.). Purine metabolism, the object of intensive research, isessential for normal functioning of the cell. Disordered purinemetabolism in higher animals may cause severe illnesses, for examplegout. Purine nucleotides are synthesized from ribose 5-phosphate by anumber of steps via the intermediate compound inosine 5′-phosphate(IMP), leading to the production of guanosine 5′-monophosphate (GMP) oradenosine 5′-monophosphate (AMP), from which the triphosphate forms usedas nucleotides can easily be prepared. These compounds are also used asenergy stores, so that breakdown thereof provides energy for manydifferent biochemical processes in the cell. Pyrimidine biosynthesistakes place via formation of uridine 5′-mono-phosphate (UMP) from ribose5-phosphate. UMP in turn is converted into cytidine 5′-triphosphate(CTP). The deoxy forms of all nucleotides are prepared in a one-stepreduction reaction from the diphosphate ribose form of the nucleotide togive the diphosphate deoxyribose form of the nucleotide. Afterphosphorylation, these molecules can take part in DNA synthesis.

D. Trehalose Metabolism and Uses

Trehalose consists of two glucose molecules linked together by □,□-1,1linkage. It is ordinarily used in the food industry as sweetener, asadditive for dried or frozen foods and in beverages. However, it is alsoused in the pharmaceutical industry or in the cosmetics industry andbiotechnology industry (see, for example, Nishimoto et al., (1998) U.S.Pat. No. 5,759,610; Singer, M. A. and Lindquist, S. Trends Biotech. 16(1998) 460-467; Paiva, C. L. A. and Panek, A. D. Biotech Ann. Rev. 2(1996) 293-314; and Shiosaka, M. J. Japan 172 (1997) 97-102). Trehaloseis produced by enzymes of many microorganisms and is naturally releasedinto the surrounding medium from which it can be isolated by methodsknown in the art.

II. Elements and Methods of the Invention

The present invention is based, at least partially, on the detection ofnew molecules which are referred to herein as G6PD nucleic-acid andG6PD-protein molecules and which are involved in taking up energy-richcarbon molecules (e.g. glucose, sucrose and fructose) into C. glutamicumand may also be involved in one or more intracellular signaltransduction pathways in this microorganism. In one embodiment, the G6PDmolecules import energy-rich carbon molecules into the cell in which theenergy generated by their degradation is used for driving energeticallyless favored biochemical reactions. Their degradation products may beused as intermediates or precursors for a number of other metabolicpathways. In another embodiment, the G6PD molecules may take part in oneor more intracellular signal transduction pathways, and the presence ofa modified form of a G6PD molecule (e.g. a phosphorylated G6PD protein)may take part in a signal transduction cascade which regulates one ormore cellular processes. In a preferred embodiment, the activity of theG6PD molecules of the invention affects the production of a finechemical of interest by said organism. In a particularly preferredembodiment, the activity of the G6PD molecules of the invention ismodulated so that the yield, production or efficiency of production ofone or more fine chemicals from C. glutamicum is likewise modulated.

In another embodiment, the G6PD molecules of the invention are capableof modulating the production of a molecule of interest, such as a finechemical, in a microorganism such as C. glutamicum. It is possible, withthe aid of gene recombination techniques, to manipulate one or more G6PDproteins of the invention in such a way that their function ismodulated. For example, a protein involved in the G6PD-mediated importof glucose may be modified for it to have optimal activity, and the G6PDsystem for importing glucose is thus able to transport larger amounts ofglucose to the cell. Glucose molecules are used not only as energysource for energetically unfavorable biochemical reactions such as thebiosynthesis of fine chemicals, but also as precursors and intermediatesin a number of biosynthetic pathways of fine chemicals (for example,serine is synthesized from 3-phosphoglycerate). In any case, it ispossible to increase the overall yield or the rate of production of anyof these fine chemicals of interest, that is by increasing the energyavailable for said production to take place or by increasing theavailability of the compounds required for said production to takeplace.

A suitable starting point for preparing the nucleic acid sequences ofthe invention is the genome of a Corynebacterium glutamicum strain whichcan be obtained from the American Type Culture Collection under the nameATCC 13032.

The nucleic acid sequences of the invention can be prepared from thesenucleic acid sequences via the modifications denoted in Table 1, usingconventional methods.

The G6PD protein of the invention or a biologically active section orfragments thereof may be involved in transporting energy-richcarbon-containing molecules such as glucose into C. glutamicum or in anintracellular signal transduction in this microorganism, or they mayhave one or more of the activities listed in Table 1.

The following subsections describe various aspects of the invention inmore detail:

A. Isolated Nucleic Acid Molecules

One aspect of the invention relates to isolated nucleic acid moleculeswhich encode G6PD polypeptides or biologically active sections thereofand to nucleic acid fragments which are sufficient for the use ashybridization probes or primers for identifying or amplifyingG6PD-encoding nucleic acids (e.g. G6PD DNA). The term “nucleic acidmolecule” is intended to comprise DNA molecules (e.g. cDNA or genomicDNA) and RNA molecules (e.g. mRNA) and also DNA or RNA analogs generatedby means of nucleotide analogs. Moreover, this term comprises theuntranslated sequence located at the 3′ and 5′ ends of the coding generegion: at least about 100 nucleotides of the sequence upstream of the5′ end of the coding region and at least about 20 nucleotides of thesequence downstream of the 3′ end of the coding gene region. The nucleicacid molecule may be single-stranded or double-stranded but ispreferably a double-stranded DNA. An “isolated” nucleic acid molecule isremoved from other nucleic acid molecules which are present in thenatural source of the nucleic acid. An “isolated” nucleic acidpreferably does not have any sequences which flank the nucleic acidnaturally in the genomic DNA of the organism from which the nucleic acidoriginates (for example sequences located at the 5′ or 3′ end of thenucleic acid). In various embodiments, the isolated G6PD nucleic acidmolecule may have, for example, less than about 5 kb, 4 kb, 3 kb, 2 kb,1 kb, 0.5 kb or 0.1 kb of the nucleotide sequences which naturally flankthe nucleic acid molecule in the genomic DNA of the cell from which thenucleic acid originates (e.g. a C. glutamicum cell). In addition tothis, an “isolated” nucleic acid molecule such as a cDNA molecule may beessentially free of another cellular material or culture medium, ifprepared by recombinant techniques, or free of chemical precursors orother chemicals, if synthesized chemically.

A nucleic acid molecule of the invention, for example a nucleic acidmolecule having a nucleotide sequence of Appendix A or a sectionthereof, may be prepared by means of molecular biological standardtechniques and the sequence information provided here. For example, a C.glutamicum G6PD cDNA may be isolated from a C. glutamicum bank by usinga complete sequence from Appendix A or a section thereof ashybridization probe and by using standard hybridization techniques (asdescribed, for example, in Sambrook, J., Fritsch, E. F. and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989). Moreover, a nucleic acid molecule comprising a completesequence from Appendix A or a section thereof can be isolated viapolymerase chain reaction, using the oligonucleotide primers produced onthe basis of said sequence (for example, it is possible to isolate anucleic acid molecule comprising a complete sequence from Appendix A ora section thereof via polymerase chain reaction by using oligonucleotideprimers which have been produced on the basis of this same sequence ofAppendix A). For example, mRNA can be isolated from normal endothelialcells (for example via the guanidinium thiocyanate extraction method ofChirgwin et al. (1979) Biochemistry 18: 5294-5299), and the cDNA can beprepared by means of reverse transcriptase (e.g. Moloney-MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md., or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for amplification viapolymerase chain reaction can be produced on the basis of any of thenucleotide sequences shown in Appendix A. A nucleic acid of theinvention may be amplified by means of cDNA or, alternatively, genomicDNA as template and of suitable oligonucleotide primers according to PCRstandard amplification techniques. The nucleic acid amplified in thisway may be cloned into a suitable vector and characterized by DNAsequence analysis. Oligonucleotides corresponding to a G6PD nucleotidesequence may be prepared by standard syntheses using, for example, anautomatic DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences listed in AppendixA.

In a further preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule complementary to any ofthe nucleotide sequences shown in Appendix A or a section thereof, saidnucleic acid molecule being sufficiently complementary to any of thenucleotide sequences shown in Appendix A for it to hybridize with any ofthe sequences indicated in Appendix A, resulting in a stable duplex.

In one embodiment, the nucleic acid molecule of the invention encodes aprotein or a section thereof comprising an amino acid sequence which issufficiently homologous to an amino acid sequence of Appendix B for theprotein or a section thereof to be still capable of taking part intransporting energy-rich carbon molecules (such as glucose) into C.glutamicum and also in one or more intracellular signal transuctionpathways. The term “sufficiently homologous”, as used herein, relates toproteins or sections thereof whose amino acid sequences have a minimumnumber of identical or equivalent amino acid residues (for example anamino acid residue having a side chain similar to that of an amino acidresidue in any of the sequences of Appendix B) compared to an amino acidsequence of Appendix B so that the protein or a section thereof iscapable of transporting energy-rich carbon molecules such as glucoseinto C. glutamicum and, moreover, taking part in the intracellularsignal transduction in this microorganism. As described herein, proteincomponents of these metabolic pathways transport energy-richcarbon-containing molecules such as glucose into C. glutamicum and mayalso be involved in intracellular signal transduction in thismicroorganism. Examples of these activities are likewise describedherein. Thus the “function of a G6PD protein” relates to the completefunctioning and/or to the regulation of one or more sugar transportpathways based on phosphoenolpyruvate. Table 1 lists examples of G6PDprotein activities.

Sections of proteins encoded by the G6PD nucleic acid molecules of theinvention are preferably biologically active sections of any of the G6PDproteins. The term “biologically active section of a G6PD protein”, asused herein, is intended to comprise a section, for example a domain ora motif, of a G6PD protein, which can transport energy-richcarbon-containing molecules such as glucose into C. glutamicum or can beinvolved in intracellular signal transduction in this microorganism, orhas an activity indicated in Table 1. In order to determine whether aG6PD protein or a biologically active section thereof can be involved intransporting energy-rich carbon-containing molecules such as glucoseinto C. glutamicum or in intracellular signal transduction in thismicroorganism, an enzyme activity assay may be carried out. These assaymethods, as described in detail in example 8 of the examples, arefamiliar to the skilled worker.

In addition to naturally occurring variants of the G6PD sequence, whichmay exist in the population, the skilled worker is likewise aware of thefact that it is possible to introduce changes into a nucleotide sequenceof Appendix A by mutation, leading to a change in the amino acidsequence of the encoded G6PD protein without impairing the functionalityof said G6PD protein. For example, it is possible to produce in asequence of Appendix A nucleotide substitutions which lead to amino acidsubstitutions at “nonessential” amino acid residues. A “nonessential”amino acid residue in a wild-type sequence of any of the G6PD proteins(Appendix B) can be modified without modifying the activity of said G6PDprotein, whereas an “essential” amino acid residue is required forG6PD-protein activity. However, other amino acid residues (e.g.nonconserved or merely semiconserved amino acid residues in the domainwith G6PD activity) may not be essential for said activity and thus canprobably be modified without modifying said G6PD activity.

An isolated nucleic acid molecule encoding a G6PD protein which ishomologous to a protein sequence of Appendix B may be generated byintroducing one or more nucleotide substitutions, additions or deletionsinto a nucleotide sequence of Appendix A so that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. The mutations may be introduced into any of the sequences ofAppendix A by standard techniques such as site-directed mutagenesis andPCR-mediated mutagenesis. Preference is given to introducingconservative amino acid substitutions at one or more of the predictednonessential amino acid residues. A “conservative amino acidsubstitution” replaces the amino acid residue by an amino acid residuewith a similar side chain. Families of amino acid residues with similarside chains have been defined in the art. These families comprise aminoacids with basic side chains (e.g. lysine, arginine, histidine), acidicside chains (e.g. aspartic acid, glutamic acid), uncharged polar sidechains (e.g. glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g. alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g. threonine, valine, isoleucine) andaromatic side chains (e.g. tyrosine, phenylalanine, tryptophan,histidine). A predicted nonessential amino acid residue in a G6PDprotein is thus preferably replaced by another amino acid residue of thesame side-chain family. In a further embodiment, the mutations mayalternatively be introduced randomly over the entire or over part of theG6PD-encoding sequence, for example by saturation mutagenesis, and theresulting mutants may be tested for the G6PD activity described hereinin order to identify mutants maintaining G6PD activity. Aftermutagenesis of any of the sequences of Appendix A, the encoded proteinmay be expressed recombinantly, and the activity of said protein may bedetermined, for example, using the assays described herein (see example8 of the examples).

B. Recombinant Expression Vectors and Host Cells

A further aspect of the invention relates to vectors, preferablyexpression vectors, containing a nucleic acid which encodes a G6PDprotein (or a section thereof). The term “vector” as used herein,relates to a nucleic acid molecule capable of transporting anothernucleic acid to which it is bound. One type of vector is a “plasmid”which term means a circular double-stranded DNA loop into whichadditional DNA segments can be ligated. Another type of vector is aviral vector, and here additional DNA segments can be ligated into theviral genome. Certain vectors are capable of replicating autonomously ina host cell into which they have been introduced (for example bacterialvectors with bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g. nonepisomal mammalian vectors) areintegrated into the genome of a host cell when introduced into said hostcell and thereby replicated together with the host genome. Moreover,particular vectors are capable of controlling the expression of genes towhich they are functionally linked. These vectors are referred to as“expression vectors”. Normally, expression vectors used in DNArecombination techniques are in the form of plasmids. In the presentdescription, “plasmid” and “vector” may be used interchangeably, sincethe plasmid is the most commonly used type of vector. The presentinvention is intended to comprise said other types of expression vectorssuch as viral vectors (for example replication-deficient retroviruses,adenoviruses and adenovirus-related viruses), which exert similarfunctions.

The recombinant expression vector of the invention comprises a nucleicacid of the invention in a form which is suitable for expressing saidnucleic acid in a host cell, meaning that the recombinant expressionvectors comprise one or more regulatory sequences which are selected onthe basis of the host cells to be used for expression and which arefunctionally linked to the nucleic acid sequence to be expressed. In arecombinant expression vector, the term “functionally linked” means thatthe nucleotide sequence of interest is bound to the regulatorysequence(s) such that expression of said nucleotide sequence is possible(for example in an in vitro transcription/translation system or in ahost cell, if the vector has been introduced into said host cell). Theterm “regulatory sequence” is intended to comprise promoters, enhancersand other expression control elements (e.g. polyadenylation signals).These regulatory sequences are described, for example, in Goeddel: GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences comprise those which controlconstitutive expression of a nucleotide sequence in many types of hostcells and those which control direct expression of the nucleotidesequence only in particular host cells. The skilled worker understandsthat designing an expression vector may depend on factors such as thechoice of host cell to be transformed, the extent of expression of theprotein of interest, etc. The expression vectors of the invention may beintroduced into the host cells so as to prepare proteins or peptides,including fusion proteins or fusion peptides, which are encoded by thenucleic acids as described herein (e.g. G6PD proteins, mutant forms ofG6PD proteins, fusion proteins, etc.).

The recombinant expression vectors of the invention may be designed forexpressing G6PD proteins in prokaryotic or eukaryotic cells. Forexample, G6PD genes may be expressed in bacterial cells such as C.glutamicum, insect cells (using baculovirus expression vectors), yeastcells and other fungal cells (see Romanos, M. A. et al. (1992) “Foreigngene expression in yeast: a review”, Yeast 8: 423-488; van den Hondel,C. A. M. J. J. et al. (1991) “Heterologous gene expression infilamentous fungi” in: More Gene Manipulations in Fungi, J. W. Bennet &L. L. Lasure, Editors, pp. 396-428: Academic Press: San Diego; and vanden Hondel, C. A. M. J. J. & Punt, P. J. (1991) “Gene transfer systemsand vector development for filamentous fungi in: Applied MolecularGenetics of Fungi, Peberdy, J. F. et al., Editors, pp. 1-28, CambridgeUniversity Press: Cambridge), algal cells and multicellular plant cells(see Schmidt, R. and Willmitzer, L. (1988) “High efficiencyAgrobacterium tumefaciens-mediated transformation of Arabidopsisthaliana leaf and cotyledon explants” Plant Cell Rep.: 583-586) ormammalian cells. Suitable host cells are further discussed in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). As an alternative, the recombinant expressionvector may be transcribed and translated in vitro, for example by usingT7 promoter regulatory sequences and T7 polymerase.

Proteins are expressed in prokaryotes mainly by using vectors containingconstitutive or inducible promoters which control expression of fusionor nonfusion proteins. Fusion vectors control a number of amino acids toa protein encoded therein, usually at the amino terminus of therecombinant protein. These fusion vectors usually have three tasks: 1)enhancing the expression of recombinant protein; 2) increasing thesolubility of the recombinant protein; and 3) supporting thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often a proteolytic cleavage site is introducedinto fusion expression vectors at the junction of fusion unit andrecombinant protein so that the recombinant protein can be separatedfrom the fusion unit after purifying the fusion protein. These enzymesand their corresponding recognition sequences comprise factor Xa,thrombin and enterokinase.

Common fusion expression vectors comprise pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT 5 (Pharmacia, Piscataway,N.J.), in which glutathione S-transferase (GST), maltose E-bindingprotein and protein A, respectively, are fused to the recombinant targetprotein. In one embodiment, the coding sequence of the G6PD protein iscloned into a pGEX expression vector such that a vector is generated,which encodes a fusion protein comprising, from N terminus to Cterminus, GST—thrombin cleavage site—protein X. The fusion protein maybe purified via affinity chromatography by means of aglutathione-agarose resin. The recombinant G6PD protein which is notfused to GST may be obtained by cleaving the fusion protein withthrombin.

Examples of suitable inducible nonfusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d(Studier et al. Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). The target geneexpression from the pTrc vector is based on transcription from a hybridtrp-lac fusion promoter by host RNA polymerase. The target geneexpression from the pET11d vector is based on transcription from aT7-gn10-lac fusion promoter, which is mediated by a coexpressed viralRNA polymerase (T7 gn1). This viral polymerase is provided in the BL 21(DE3) or HMS174 (DE3) host strain by a resident

prophage which harbors a T7 gn1 gene under the transcriptional controlof the lacUV 5 promoter.

One strategy for maximizing expression of the recombinant protein is toexpress said protein in a host bacterium whose ability toproteolytically cleave said recombinant protein is disrupted (Gottesman,S. Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Another strategy is to modifythe nucleic acid sequence of the nucleic acid to be inserted into anexpression vector such that the individual codons for each amino acidare those which are preferably used in a bacterium selected forexpression, such as C. glutamicum (Wada et al. (1992) Nucleic Acids Res.20: 2111-2118). This modification of the nucleic acid sequences of theinvention is carried out by standard techniques of DNA synthesis.

In a further embodiment, the G6PD-protein expression vector is a yeastexpression vector. Examples of vectors for expression in the yeast S.cerevisiae include pYepSec1 (Baldari et al., (1987) Embo J. 6: 229-234),pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz etal. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, SanDiego, Calif.). Vectors and methods for constructing vectors which aresuitable for use in other fungi such as filamentous fungi include thosewhich are described in detail in: van den Hondel, C. A. M. J. J. & Punt,P. J. (1991) “Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of fungi, J. F.Peberdy et al., Editors, pp. 1-28, Cambridge University Press:Cambridge.

As another alternative, it is possible to express the G6PD proteins ofthe invention in insect cells using baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g. Sf9 cells) include the pAc series (Smith et al.,(1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series (Lucklow andSummers (1989) Virology 170: 31-39).

In a further embodiment, the G6PD proteins of the invention may beexpressed in unicellular plant cells (such as algae) or in cells of thehigher plants (e.g. spermatophytes such as crops). Examples ofexpression vectors of plants include those which are described in detailin: Bekker, D., Kemper, E., Schell, J. and Masterson, R. (1992) “Newplant binary vectors with selectable markers located proximal to theleft border”, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W. (1984)“Binary Agrobacterium vectors for plant transformation”, Nuci. AcidsRes. 12: 8711-8721.

A further embodiment, a nucleic acid of the invention is expressed inmammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195). Whenused in mammalian cells, the control functions of the expression vectorare often provided by viral regulatory elements. Commonly used promotersare derived, for example, from polyoma, adenovirus2, cytomegalovirus andsimian virus 40. Other suitable expression systems for prokaryotic andeukaryotic cells can be found in chapters 16 and 17 of Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular cloning: A Laboratory Manual,2nd Edition, Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

In a further embodiment, the recombinant mammalian expression vector maypreferably cause expression of the nucleic acid in a particular celltype (for example, tissue-specific regulatory elements are used forexpressing the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43: 235-275), in particular promoters ofT-cell receptors (Winoto and Baltimore (1989) EMBO J. 8: 729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33: 729-740; Queen andBaltimore (1983) Cell 33: 741-748), neuron-specific promoters (e.g.neurofilament promoter; Byrne and Ruddle (1989) PNAS 86: 5473-5477),pancreas-specific promoters (Edlund et al., (1985) Science 230: 912-916)and mamma-specific promoters (e.g. milk serum promoter; U.S. Pat. No.4,873,316 and European Patent Application document No. 264 166).Development-regulated promoters for example the murine hox promoters(Kessel and Gruss (1990) Science 249: 374-379) and the □-fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3: 537-546), arelikewise included.

Moreover, the invention provides a recombinant expression vectorcomprising an inventive DNA molecule which has been cloned into theexpression vector in antisense direction. This means that the DNAmolecule is functionally linked to a regulator sequence such that an RNAmolecule which is antisense to G6PD mRNA can be expressed (viatranscription of the DNA molecule). It is possible to select regulatorysequences which are functionally bound to a nucleic acid cloned inantisense direction and which control continuous expression of theantisense RNA molecule in a multiplicity of cell types; for example, itis possible to select viral promoters and/or enhancers or regulatorysequences which control the constitutive tissue-specific or celltype-specific expression of antisense RNA. The antisense expressionvector may be in the form of a recombinant plasmid, phagemid orattenuated virus and produces antisense nucleic acids under the controlof a highly effective regulatory region whose activity is determined bythe cell type into which the vector is introduced. The regulation ofgene expression by means of antisense genes is discussed in Weintraub,H. et al., Antisense-RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

A further aspect of the invention relates to the host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. Naturally, these terms relate not only to a particular targetcell but also to the progeny or potential progeny of this cell. Sinceparticular modifications may appear in successive generations, due tomutation or environmental factors, this progeny is not necessarilyidentical to the parental cell but is still included within the scope ofthe term as used herein.

A host cell may be a prokaryotic or eukaryotic cell. For example, a G6PDprotein may be expressed in bacterial cells such as C. glutamicum,insect cells, yeast cells or mammalian cells (such as Chinese hamsterovary (CHO) cells or COS cells). Other suitable host cells are familiarto the skilled worker. Microorganisms which are related toCorynebacterium glutamicum and can be used in a suitable manner as hostcells for the nucleic acid and protein molecules of the invention arelisted in Table 3.

Conventional transformation or transfection methods can be used tointroduce vector DNA into prokaryotic or eukaryotic cells. The terms“transformation” and “transfection”, as used herein, are intended tocomprise a multiplicity of methods known in the art for introducingforeign nucleic acid (e.g. DNA) into a host cell, including calciumphosphate or calcium chloride coprecipitation, DEAE dextran-mediatedtransfection, lipofection or electroporation. Suitable methods fortransformation or transfection of host cells can be found in Sambrook etal. (Molecular Cloning: A Laboratory Manual. 2nd Edition, Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989) and other laboratory manuals.

In the case of stable transfection of mammalian cells, it is known that,depending on the expression vector used and transfection technique used,only a small proportion of the cells integrate the foreign DNA intotheir genome. These integrants are usually identified and selected byintroducing a gene which encodes a selectable marker (e.g. resistant toantibiotics) together with the gene of interest into the host cells.Preferred selectable markers include those which impart resistance todrugs such as G418, hygromycin and methotrexate. A nucleic acid whichencodes a selectable marker may be introduced into a host cell on thesame vector that encodes a G6PD protein or may be introduced in aseparate vector. Cells which have been stably transfected with theintroduced nucleic acid may be identified by drug selection (forexample, cells which have integrated the selectable marker survive,whereas the other cells die).

A homologous recombined microorganism is generated by preparing a vectorwhich contains at least one G6PD-gene section into which a deletion,addition or substitution has been introduced in order to modify, forexample functionally disrupt, the G6PD gene. Said G6PD gene ispreferably a Corynebacterium glutamicum G6PD gene, but it is alsopossible to use a homolog from a related bacterium or even from amammalian, yeast or insect source. In a preferred embodiment, the vectoris designed such that homologous recombination functionally disrupts theendogenous G6PD gene (i.e., the gene no longer encodes a functionalprotein; likewise referred to as “knockout” vector). As an alternative,the vector may be designed such that homologous recombination mutates orotherwise modifies the endogenous G6PD gene which, however, stillencodes the functional protein (for example, the regulatory regionlocated upstream may be modified such that thereby expression of theendogenous G6PD protein is modified.). The modified G6PD-gene fractionin the homologous recombination vector is flanked at its 5′ and 3′ endsby additional nucleic acids of the G6PD gene, which makes possible ahomologous recombination between the exogenous G6PD gene carried by thevector and an endogenous G6PD gene in a microorganism. The length of theadditional flanking G6PD nucleic acid is sufficient for a successfulhomologous recombination with the endogenous gene. Usually, the vectorcontains several kilobases of flanking DNA (both at the 5′ and the 3′ends) (see, for example, Thomas, K. R. and Capecchi, M. R. (1987) Cell51: 503, for a description of homologous recombination vectors). Thevector is introduced into a microorganism (e.g. by electroporation) andcells in which the introduced G6PD gene has homologously recombined withthe endogenous G6PD gene are selected using methods known in the art.

In another embodiment, it is possible to produce recombinantmicroorganisms which contain selected systems which make possible aregulated expression of the introduced gene. The insertion of a G6PDgene under the control of the lac operon in a vector enables, forexample, G6PD-gene expression only in the presence of IPTG. Theseregulatory systems are known in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, may be used for producing (i.e. expressing) a G6PDprotein. Moreover, the invention provides methods for producing G6PDproteins by using the host cells of the invention. In one embodiment,the method comprises the cultivation of the host cell of the invention(into which a recombinant expression vector encoding a G6PD protein hasbeen introduced or in whose genome a gene encoding a wild-type ormodified G6PD protein has been introduced) in a suitable medium untilthe G6PD protein has been produced. In a further embodiment, the methodcomprises isolating the G6PD proteins from the medium or the host cell.

C. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologs, fusion proteins,primers, vectors and host cells described herein may be used in one ormore of the following methods: identification of C. glutamicum andrelated organisms, mapping of genomes of organisms related to C.glutamicum, identification and localization of C. glutamicum sequencesof interest, evolutionary studies, determination of G6PD-protein regionsrequired for function, modulation of the activity of G6PD protein;modulation of the activity of a G6PD pathway; and modulation of thecellular production of a compound of interest, such as a fine chemical.The G6PD nucleic acid molecules of the invention have a multiplicity ofuses. First, they may be used for identifying an organism asCorynebacterium glutamicum or close relatives thereof. They may also beused for identifying C. glutamicum or a relative thereof in a mixedpopulation of microorganisms. The invention provides the nucleic acidsequences of a number of C. glutamicum genes. Probing the extractedgenomic DNA of a culture of a uniform or mixed population ofmicroorganisms under stringent conditions with a probe which comprises aregion of a C. glutamicum gene which is unique for this organism makesit possible to determine whether said organism is present. AlthoughCorynebacterium glutamicum itself is nonpathogenic, it is related topathogenic species such as Corynebacterium diphtheriae. The detection ofsuch an organism is of substantial clinical importance.

The nucleic acid and protein molecules of the invention may serve asmarkers for specific regions of the genome. This is useful not only formapping the genome but also for functional studies of C. glutamicumproteins. The genomic region to which a particular C. glutamicumDNA-binding protein binds may be identified, for example, by cleavingthe C. glutamicum genome and incubating the fragments with theDNA-binding protein. Those fragments which bind the protein mayadditionally be probed with the nucleic acid molecules of the invention,preferably by using ready detectable labels; binding of such a nucleicacid molecule to the genomic fragment makes it possible to locate thefragment on the map of the C. glutamicum genome, and, when carrying outthe process several times using different enzymes, facilitates rapiddetermination of the nucleic acid sequence to which the protein binds.Moreover, the nucleic acid molecules of the invention may besufficiently homologous to the sequences of related species for thesenucleic acid molecules to serve as markers for constructing a genomicmap in related bacteria such as Brevibacterium lactofermentum.

The G6PD nucleic acid molecules of the invention are likewise suitablefor evolutionary studies and protein structure studies. The system fortaking up sugars, in which the molecules of the invention are involved,is utilized by many bacteria; comparison of the sequences of the nucleicacid molecules of the invention with those encoding similar enzymes ofother organisms makes it possible to determine the degree ofevolutionary relationship of said organisms. Correspondingly, such acomparison makes it possible to determine which sequence regions areconserved and which are not, and this can be helpful in determiningthose regions of the protein, which are essential for enzyme function.This type of determination is valuable for protein engineering studiesand may give an indication as to which protein can tolerate mutagenesiswithout losing its function.

Manipulation of the G6PD nucleic acid molecules of the invention maycause the production of G6PD proteins with functional differences towild-type G6PD proteins. These proteins may be improved with respect totheir efficiency or activity, may be present in the cell in largeramounts than normal or may be weakened with respect to their efficiencyor activity.

The G6PD molecules of the invention may be modified in such a way thatthe yield, production and/or efficiency of production of one or morefine chemicals is improved. It is possible, by modifying a G6PD proteininvolved in taking up glucose for it to have optimal activity, toincrease the extent of glucose uptake or the rate at which glucose istransported into the cell. The degradation of glucose and other sugarsinside the cell provides the energy which may be used for drivingenergetically unfavorable biochemical reactions such as those involvedin the biosynthesis of fine chemicals. Said degradation likewiseprovides intermediates and precursors for the biosynthesis of particularfine chemicals such as amino acids, vitamins and cofactors. It istherefore possible, by increasing the amount of intracellularenergy-rich carbon molecules via modification of the G6PD molecules ofthe invention, to increase the energy available for carrying outmetabolic pathways which are required for producing one or more finechemicals and also the intracellular pools of metabolites required forsaid production. Conversely, it is possible, by lowering the import of asugar whose degradation products include a compound which is used onlyin metabolic pathways competing for enzymes, cofactors or intermediateswith metabolic pathways for producing a fine chemical of interest, todownregulate said metabolic pathway and thus perhaps increase productionby the biosynthetic pathway of interest.

The G6PD molecules of the invention may be involved in one or moreintracellular signal transduction pathways which influence the yieldsand/or rate of production of one or more fine chemicals from C.glutamicum. For example, proteins required for importing one or moresugars from the extracellular medium (e.g. HPr, enzyme I or a componentof an enzyme II complex) are, in the presence of a sufficient amount ofsaid sugar in the cell, frequently posttranslationally modified so thatthey are no longer capable of importing said sugar. An example of thistakes place in E. coli in which high intracellular fructose1,6-bisphosphate levels cause phosphorylation of HPr at serine 46, afterwhich said molecule can no longer take part in transporting a sugar.Although this intracellular sugar level at which the transport systsemis switched off may be sufficient in order to maintain normal cellularfunction, it limits overproduction of the fine chemical of interest. Itis therefore desirable to modify the G6PD proteins of the invention suchthat they are no longer susceptible to such a negative regulation. As aresult, higher intracellular concentrations of one or more sugars and,by extension, a more efficient production or higher yields of one ormore fine chemicals from organisms containing these mutant G6PD proteinsare attained.

This abovementioned list of strategies for the mutagenesis of G6PDproteins, which ought to increase the yields of a compound of interest,is not intended to be limiting; variations of these mutagenesisstrategies are quite obvious to the skilled worker. These mechanismsmake it possible to use the nucleic acid and protein molecules of theinvention in order to generate C. glutamicum or related bacterialstrains expressing mutated G6PD nucleic acids and protein molecules soas to improve the yield, production and/or efficiency of production of acompound of interest. The compound of interest may be a C. glutamicumproduct which comprises the end products of the biosynthetic pathwaysand intermediates of naturally occurring metabolic pathways and alsomolecules which do not naturally occur in the C. glutamicum metabolismbut are produced by a C. glutamicum strain of the invention.

The following examples which are not to be understood as being limitingfurther illustrate the present invention. The contents of allreferences, patent applications, patents and published patentapplications cited in this patent application are hereby incorporated byway of reference.

EXAMPLES Example 1 Preparation of Total Genomic DNA from Corynebacteriumglutamicum ATCC13032

A Corynebacterium glutamicum (ATCC 13032) culture was cultivated withvigorous shaking in BHI medium (Difco) at 30_C overnight. The cells wereharvested by centrifugation, the supernatant was discarded and the cellswere resuspended in 5 ml of buffer I (5% of the original culturevolume—all volumes stated have been calculated for a culture volume of100 ml). Composition of buffer I: 140.34 g/l sucrose, 2.46 g/l MgSO₄ □ 7H₂O, 10 ml/l KH₂PO₄ solution (100 g/l, adjusted to pH 6.7 with KOH), 50ml/l M12 concentrate (10 g/l (NH₄)₂SO₄, 1 g/l NaCl, 2 g/l MgSO₄ □ 7 H₂O,0.2 g/l CaCl₂, 0.5 g/l yeast extract (Difco), 10 ml/l trace elementmixture (200 mg/l FeSO₄ □ H₂O, 10 mg/l ZnSO₄ □ 7 H₂O, 3 mg/l MnCl₂ □ 4H₂O, 30 mg/l H₃BO₃, 20 mg/l CoCl₂ □ 6 H₂O, 1 mg/l NiCl₂ □ 6 H₂O, 3 mg/lNa₂MoO₄ □ 2 H₂O), 500 mg/l complexing agents (EDTA or citric acid), 100ml/l vitamin mixture (0.2 ml/l biotin, 0.2 mg/l folic acid, 20 mg/lp-aminobenzoic acid, 20 mg/l riboflavin, 40 mg/l Ca panthothenate, 140mg/l nicotinic acid, 40 mg/l pyridoxal hydrochloride, 200 mg/lmyoinositol). Lysozyme was added to the suspension at a finalconcentration of 2.5 mg/ml. After incubation at 37_C for approx. 4 h,the cell wall was degraded and the protoplasts obtained were harvestedby centrifugation. The pellet was washed once with 5 ml of buffer I andonce with 5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). Thepellet was resuspended in 4 ml of TE buffer and 0.5 ml of SDS solution(10%) and 0.5 ml of NaCl solution (5 M) were added. After addition ofproteinase K at a final concentration of 200 □g/ml, the suspension wasincubated at 37_C for approx. 18 h. The DNA was purified via extractionwith phenol, phenol/chloroform/isoamyl alcohol and chloroform/isoamylalcohol by means of standard methods. The DNA was then precipitated byaddition of 1/50 volume of 3 M sodium acetate and 2 volumes of ethanol,subsequent incubation at −20_C for 30 min and centrifugation at 12 000rpm in a high-speed centrifuge using an SS34 rotor (Sorvall) for 30 min.The DNA was dissolved in 1 ml of TE buffer containing 20 □/g/ml RNase Aand dialyzed against 1000 ml of TE buffer at 4_C for at least 3 h. Thebuffer was exchanged 3 times during this period. 0.4 ml of 2 M LiCl and0.8 ml of ethanol were added to 0.4 ml aliquots of the dialyzed DNAsolution. After incubation at −20_C for 30 min, the DNA was collected bycentrifugation (13 000 rpm, Biofuge Fresco, Heraeus, Hanau, Germany).The DNA pellet was dissolved in TE buffer. It was possible to use DNAprepared by this method for all purposes, including Southern blottingand constructing genomic libraries.

Example 2 Construction of Genomic Corynebacterium glutamicwn (ATCC13032)Banks in Escherichia coli

Starting from DNA prepared as described in Example 1, cosmid and plasmidbanks were prepared according to known and well-established methods(see, for example, Sambrook, J. et al. (1989) “Molecular Cloning: ALaboratory Manual”. Cold Spring Harbor Laboratory Press or Ausubel, F.M. et al. (1994) “Current Protocols in Molecular Biology”, John Wiley &Sons).

It was possible to use any plasmid or cosmid. Particular preference wasgiven to using the plasmids pBR322 (Sutcliffe, J. G. (1979) Proc. NatlAcad. Sci. USA, 75: 3737-3741); pACYC177 (Change & Cohen (1978) J.Bacteriol. 134: 1141-1156); pBS series plasmids (pBSSK+, pBSSK− andothers; Stratagene, LaJolla, USA) or cosmids such as SuperCos1(Stratagene, LaJolla, USA) or Lorist6 (Gibson, T. J. Rosenthal, A., andWaterson, R. H. (1987) Gene 53: 283-286.

Example 3 DNA Sequencing and Functional Computer Analysis

Genomic banks, as described in Example 2, were used for DNA sequencingaccording to standard methods, in particular the chain terminationmethod using ABI377 sequencers (see, for example, Fleischman, R. D. etal. (1995) “Whole-genome Random Sequencing and Assembly of HaemophilusInfluenzae Rd., Science 269; 496-512). Sequencing primers having thefollowing nucleotide sequences were used: 5′-GGAAACAGTATGACCATG-3′ oder5′-GTAAAACGACGGCCAGT-3′.

Example 4 In Vivo Mutagenesis

In vivo mutagenesis of Corynebacterium glutamicum may be carried out bypassing a plasmid (or other vector) DNA through E. coli or othermicroorganisms (e.g. Bacillus spp. or yeasts such as Saccharomycescerevisiae) which cannot maintain the integrity of their geneticinformation. Common mutator strains contain mutations in the genes forthe DNA repair system (e.g., mutHLS, mutD, mutT, etc., for comparisonsee Rupp, W. D. (1996) DNA repair mechanisms in Escherichia coli andSalmonella, pp. 2277-2294, ASM: Washington). These strains are known tothe skilled worker. The use of these strains is illustrated, forexample, in Greener, A. and Callahan, M. (1994) Strategies 7; 32-34.

Example 5 DNA Transfer Between Escherichia coli and Corynebacteriumglutamicum

A plurality of Corynebacterium and Brevibacterium species containendogenous plasmids (such as, for example, pHM1519 or pBL1) whichreplicate autonomously (for a review see, for example, Martin, J. F. etal. (1987) Biotechnology 5: 137-146). Shuttle vectors for Escherichiacoli and Corynebacterium glutamicum can be constructed readily by meansof standard vectors for E. coli (Sambrook, J. et al., (1989), “MolecularCloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press orAusubel, F. M. et al. (1994) “Current Protocols in Molecular Biology”,John Wiley & Sons), to which an origin of replication for and a suitablemarker from Corynebacterium glutamicum is added. Such origins ofreplication are preferably taken from endogenous plasmids which havebeen isolated from Corynebacterium and Brevibacterium species.Particularly useful transformation markers for these species are genesfor kanamycin resistance (such as those derived from the Tn5 or theTn903 transposon) or for chloramphenicol resistance (Winnacker, E. L.(1987) “From Genes to Clones—Introduction to Gene Technology, VCH,Weinheim). There are numerous examples in the literature for preparing alarge multiplicity of shuttle vectors which are replicated in E. coliand C. glutamicum and which can be used for various purposes, includingthe overexpression of genes (see, for example, Yoshihama, M. et al.(1985) J. Bacteriol. 162: 591-597, Martin, J. F. et al., (1987)Biotechnology, 5: 137-146 and Eikmanns, B. J. et al. (1992) Gene 102:93-98).

Standard methods make it possible to clone a gene of interest into oneof the above-described shuttle vectors and to introduce such hybridvectors into Corynebacterium glutamicum strains. C. glutamicum can betransformed via protoplast transformation (Kastsumata, R. et al., (1984)J. Bacteriol. 159, 306-311), electroporation (Liebl, E. et al., (1989)FEMS Microbiol. Letters, 53: 399-303) and, in cases in which specificvectors are used, also via conjugation (as described, for example, inSchäfer, A., et al. (1990) J. Bacteriol. 172: 1663-1666). Likewise, itis possible to transfer the shuttle vectors for C. glutamicum to E. coliby preparing plasmid DNA from C. glutamicum (by means of standardmethods known in the art) and transforming it into E. coli. Thistransformation step can be carried out using standard methods butadvantageously an Mcr-deficient E. coli strain such as NM522 (Gough &Murray (1983) J. Mol. Biol. 166: 1-19) is used.

Example 6 Determination of the Expression of the Mutated Protein

The observations of the activity of a mutated protein in a transformedhost cell are based on the fact that the mutated protein is expressed ina similar manner and in similar quantity to the wild-type protein. Asuitable method for determining the amount of transcription of themutated gene (an indication of the amount of mRNA available fortranslation of the gene product) is to carry out a Northern blot (see,for example, Ausubel et al., (1988) Current Protocols in MolecularBiology, Wiley: N.Y.), with a primer which is designed such that itbinds to the gene of interest being provided with a detectable (usuallyradioactive or chemiluminescent) label such that—when the total RNA of aculture of the organism is extracted, fractionated on a gel, transferredto a stable matrix and incubated with this probe—binding and bindingquantity of the probe indicate the presence and also the amount of mRNAfor said gene. This information is an indicator of the extent to whichthe mutant gene has been transcribed. Total cell RNA can be isolatedfrom Corynebacterium glutanficum by various methods known in the art, asdescribed in Bormann, E R et al., (1992) Mol. Microbiol. 6: 317-326.

The presence or the relative amount of protein translated from said mRNAcan be determined by using standard techniques such as Western blot(see, for example, Ausubel et al. (1988) “Current Protocols in MolecularBiology”, Wiley, N.Y.). In this method, total cell proteins areextracted, separated by gel electrophoresis, transferred to a matrixsuch as nitrocellulose and incubated with a probe, for example anantibody, which binds specifically to the protein of interest. Thisprobe is usually provided with a chemiluminescent or colorimetric labelwhich can be readily detected. The presence and the observed amount oflabel indicate the presence and the amount of the desired mutant proteinin the cell.

Example 7 Growth of Genetically Modified Corynebacteriumglutamicum—Media and Cultivation Conditions

Genetically modified corynebacteria are cultivated in synthetic ornatural growth media. A number of different growth media forcorynebacteria are known and readily available (Lieb et al. (1989) Appl.Microbiol. Biotechnol. 32: 205-210; von der Osten et al. (1998)Biotechnology Letters 11: 11-16; Patent DE 4 120 867; Liebl (1992) “TheGenus Corynebacterium”, in: The Procaryotes, Vol. II, Balows, A., etal., editors Springer-Verlag). These media are composed of one or morecarbon sources, nitrogen sources, inorganic salts, vitamins and traceelements. Preferred carbon sources are sugars such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch and cellulose. Sugars may also beadded to the media via complex compounds such as molasses or otherbyproducts from sugar refining It may also be advantageous to addmixtures of various carbon sources. Other possible carbon sources arealcohols and organic acids, such as methanol, ethanol, acetic acid orlactic acid. Nitrogen sources are usually organic or inorganic nitrogencompounds or materials containing these compounds. Examples of nitrogensources include ammonia gas and ammonium salts such as NH₄Cl or(NH₄)₂SO₄, NH₄OH, nitrates, urea, amino acids and complex nitrogensources such as cornsteep liquor, soya meal, soya protein, yeastextracts, meat extracts and others.

Inorganic salt compounds which may be present in the media include thechloride, phosphorus or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents includedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid. The media usually also contain other growth factorssuch as vitamins or growth promoters, examples of which include biotin,riboflavin, thiamine, folic acid, nicotinic acid, panthothenate andpyridoxine. Growth factors and salts are frequently derived from complexmedia components such as yeast extract, molasses, cornsteep liquor andthe like. The exact composition of the media heavily depends on theparticular experiment and is decided upon individually for each case.Information on the optimization of media can be found in the textbook“Applied Microbiol. Physiology, A Practical Approach” (editors P. M.Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3).Growth media can also be obtained from commercial suppliers, for exampleStandard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121_C) or by sterile filtration. The components may be sterilizedeither together or, if required, separately. All media components may bepresent at the start of the cultivation or added continuously orbatchwise, as desired.

The cultivation conditions are defined separately for each experiment.The temperature should be between 15_C and 45_C and may be kept constantor may be altered during the experiment. The pH of the medium should bein the range from 5 to 8.5, preferably around 7.0 and may be maintainedby adding buffers to the media. An example of a buffer for this purposeis a potassium phosphate buffer. Synthetic buffers such as MOPS, HEPES;ACES, etc. may be used alternatively or simultaneously. Addition of NaOHor NH₄OH can also keep the pH constant during cultivation. If complexmedia components such as yeast extract are used, the demand foradditional buffers decreases, since many complex compounds have a highbuffer capacity. In the case of using a fermenter for cultivatingmicroorganisms, the pH may also be regulated using gaseous ammonia.

The incubation period is usually in a range from several hours toseveral days. This time is selected such that the maximum amount ofproduct accumulates in the broth. The growth experiments disclosed maybe carried out in a multiplicity of containers such as microtiterplates, glass tubes, glass flasks or glass or metal fermenters ofdifferent sizes. For the screening of a large number of clones, themicroorganisms should be grown in microtiter plates, glass tubes orshaker flasks either with or without baffles. Preference is given tousing 100 ml shaker flasks which are filled with 10% (based on volume)of the required growth medium. The flasks should be shaken on an orbitalshaker (amplitude 25 mm) at a speed in the range of 100-300 rpm. Lossesdue to evaporation can be reduced by maintaining a humid atmosphere;alternatively, the losses due to evaporation should be correctedmathematically.

If genetically modified clones are investigated, an unmodified controlclone or a control clone containing the basic plasmid without insertshould also be assayed. The medium is inoculated to an OD₆₀₀ of 0.5-1.5,with cells being used which have been grown on agar plates such as CMplates (10 g/l glucose, 2.5 g/l NaCl, 2 g/l urea, 10 g/l polypeptone, 5g/l yeast extract, 5 g/l meat extract, 22 g/l agar pH 6.8 with 2 M NaOH)which have been incubated at 30_C. The media are inoculated either byintroducing a saline solution of C. glutamicum cells from CM plates orby adding a liquid preculture of said bacterium.

Example 8 In Vitro Analysis of the Function of Mutated Proteins

The determination of the activities and kinetic parameters of enzymes iswell known in the art. Experiments for determining the activity of aparticular modified enzyme must be adapted to the specific activity ofthe wild-type enzyme, and this is within the capabilities of the skilledworker. Overviews regarding enzymes in general and also specific detailsconcerning the structure, kinetics, principles, methods, applicationsand examples of the determination of many enzyme activities can befound, for example, in the following references: Dixon, M., and Webb, E.C: (1979) Enzymes, Longmans, London; Fersht (1985) Enzyme Structure andMechanism, Freeman, New York; Walsh (1979) Enzymatic ReactionMechanisms. Freeman, San Francisco; Price, N. C., Stevens, L. (1982)Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D:editors (1983) The Enzymes, 3rd edition, Academic Press, New York;Bisswanger, H. (1994) Enzymkinetik, 2nd edition VCH, Weinheim (ISBN3527300325); Bergmeyer, H. U., Bergmeyer, J., Graβl, M. editors(1983-1986) Methods of Enzymatic Analysis, 3rd edition, Vol. I-XII,Verlag Chemie: Weinheim; and Ullmann's Encyclopedia of IndustrialChemistry (1987) Vol. A9, “Enzymes”, VCH, Weinheim, pp. 352-363.

The activity of proteins binding to DNA can be measured by manywell-established methods such as DNA bandshift assays (which are alsoreferred to as gel retardation assays). The action of these proteins onthe expression of other molecules can be measured using reporter geneassays (as described in Kolmar, H. et al., (1995) EMBO J. 14: 3895-3904and in references therein). Reporter gene assay systems are well knownand established for applications in prokaryotic and eukaryotic cells,with enzymes such as beta-galactosidase, green fluorescent protein andseveral other enzymes being used.

The activity of membrane transport proteins can be determined accordingto the techniques described in Gennis, R. B. (1989) “Pores, Channels andTransporters”, in Biomembranes, Molecular Structure and Function,Springer: Heidelberg, pp. 85-137; 199-234; and 270-322.

Example 9 Analysis of the Influence of Mutated Protein on the Productionof the Product of Interest

The effect of the genetic modification in C. glutamicum on theproduction of a compound of interest (such as an amino acid) can bedetermined by growing the modified microorganisms under suitableconditions (such as those described above) and testing the medium and/orthe cellular components for increased production of the product ofinterest (i.e. an amino acid). Such analytical techniques are well knownto the skilled worker and include spectroscopy, thin-layerchromatography, various types of coloring methods, enzymic andmicrobiological methods and analytical chromatography such as highperformance liquid chromatography (see, for example, Ullman,Encyclopedia of Industrial Chemistry, Vol. A2, pp. 89-90 and pp.443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) “Applicationsof HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 17; Rehm et al. (1993) Biotechnology, Vol. 3,Chapter III: “Product recovery and purification”, pp. 469-714, VCH:Weinheim; Belter, P. A. et al. (1988) Bioseparations: downstreamprocessing for Biotechnology, John Wiley and Sons; Kennedy, J. F. andCabral, J. M. S. (1992) Recovery processes for biological Materials,John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D. (1988)Biochemical Separations, in Ullmann's Encyclopedia of IndustrialChemistry, Vol. B3; Chapter 11, pp. 1-27, VCH: Weinheim; and Dechow, F.J. (1989) Separation and purification techniques in biotechnology, NoyesPublications).

In addition to measuring the end product of the fermentation, it islikewise possible to analyze other components of the metabolic pathways,which are used for producing the compound of interest, such asintermediates and byproducts, in order to determine the overallproductivity of the organism, the yield and/or the efficiency ofproduction of the compound. The analytical methods include measuring theamounts of nutrients in the medium (for example sugars, hydrocarbons,nitrogen sources, phosphate and other ions), measuring biomasscomposition and growth, analyzing the production of common metabolitesfrom biosynthetic pathways and measuring gases generated duringfermentation. Standard methods for these measurements are described inApplied Microbial Physiology; A Practical Approach, P. M. Rhodes and P.F. Stanbury, editors IRL Press, pp. 103-129; 131-163 and 165-192 (ISBN:0199635773) and the references therein.

Example 10 Purification of the Product of Interest from a C. glutamicumCulture

The product of interest may be obtained from C. glutamicum cells or fromthe supernatant of the above-described culture by various methods knownin the art. If the product of interest is not secreted by the cells, thecells may be harvested from the culture by slow centrifugation, and thecells may be lysed by standard techniques such as mechanial force orsonication. The cell debris is removed by centrifugation and thesupernatant fraction which contains the soluble proteins is obtained forfurther purification of the compound of interest. If the product issecreted by the C. glutamicum cells, the cells are removed from theculture by slow centrifugation and the supernatant fraction is retainedfor further purification.

The supernatant fraction from both purification methods is subjected tochromatography using a suitable resin, and either the molecule ofinterest is retained on the chromatography resin while many contaminantsin the sample are not, or the contaminants remain on the resin while thesample does not. If necessary, these chromatography steps can berepeated using the same or different chromatography resins. The skilledworker is familiar with the selection of suitable chromatography resinsand the most effective application for a particular molecule to bepurified. The purified product may be concentrated by filtration orultrafiltration and stored at a temperature at which product stabilityis highest.

In the art, many purification methods are known which are not limited tothe above purification method and which are described, for example, inBailey, J. E. & Ollis, D. F. Biochemical Engineering Fundamentals,McGraw-Hill: New York (1986).

The identity and purity of the isolated compounds can be determined bystandard techniques of the art. These techniques comprise highperformance liquid chromatography (HPLC), spectroscopic methods,coloring methods, thin-layer chromatography, NIRS, enzyme assays ormicrobiological assays. These analytical methods are compiled in: Pateket al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al.(1996) Biotekhnologiya 11: 27-32; and Schmidt et al. (1998) BioprocessEngineer. 19: 67-70. Ulmann's Encyclopedia of Industrial Chemistry(1996) Vol. A27, VCH: Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp.559-566, 575-581 and pp. 581-587; Michal, G (1999) Biochemical Pathways:An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons;Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 17.

Equivalents

The skilled worker knows, or can identify by using simply routinemethods, a large number of equivalents of the specific embodiments ofthe invention. These equivalents are intended to be included in thepatent claims below.

The information in Table 1 is to be understood as follows:

In column 1, “DNA ID”, the relevant number refers in each case to theSEQ ID NO of the enclosed sequence listing. Consequently, “5” in column“DNA ID” is a reference to SEQ ID NO:5.

In column 2, “AA ID”, the relevant number refers in each case to the SEQID NO of the enclosed sequence listing. Consequently, “6” in column “AAID” is a reference to SEQ ID NO:6.

In column 3, “Identification”, an unambiguous internal name for eachsequence is listed.

In column 4, “AA pos”, the relevant number refers in each case to theamino acid position of the polypeptide sequence “AA ID” in the same row.Consequently, “26” in column “AA pos” is amino acid position 26 of thepolypeptide sequence indicated accordingly. Position counting starts atthe N terminus with +1.

In column 5, “AA wild type”, the relevant letter refers in each case tothe amino acid, displayed in the one-letter code, at the position in thecorresponding wild-type strain, which is indicated in column 4.

In column 6, “AA mutant”, the relevant letter refers in each case to theamino acid, displayed in the one-letter code, at the position in thecorresponding mutant strain, which is indicated in column 4.

In column 7, “Function”, the physiological function of the correspondingpolypeptide sequence is listed.

One-Letter Code of the Proteinogenic Amino Acids:

-   A Alanine-   C Cysteine-   D Aspartic acid-   E Glutamic acid-   F Phenylalanine-   G Glycine-   H Histidine-   I Isoleucine-   K Lysine-   L Leucine-   M Methionine-   N Asparagine-   P Proline-   Q Glutamine-   R Arginine-   S Serine-   T Threonine-   V Valine-   W Tryptophan-   Y Tyrosine

TABLE 1 Genes coding for glucose-6-phosphate-dehydrogenase proteins AADNA AA AA wild AA ID: ID: Identification: pos: type mutant Function: 1 2RXA02737 243 A T Glucose-6- phosphate dehydrogenase

1. An isolated nucleic acid molecule, coding for a polypeptide havingthe amino acid sequence referred to in each case in Table 1/column 2,wherein the nucleic acid molecule in the amino acid position indicatedin table 1/column 4 encodes a proteinogenic amino acid different fromthe particular amino acid indicated in Table 1/column 5 in the same row.2. An isolated nucleic acid molecule as claimed in claim 1, wherein thenucleic acid molecule in the amino acid position indicated in Table1/column 4 encodes the amino acid indicated in Table 1/column 6 in thesame row.
 3. A vector, which comprises at least one nucleic acidsequence as claimed in claim
 1. 4. A host cell, which is transfectedwith at least one vector as claimed in claim
 3. 5. A host cell asclaimed in claim 4, wherein expression of said nucleic acid moleculemodulates the production of a fine chemical from said cell.
 6. A methodfor preparing a fine chemical, which comprises culturing a cell whichhas been transfected with at least one vector as claimed in claim 3 sothat the fine chemical is produced.
 7. A method as claimed in claim 6,wherein the fine chemical is an amino acid.
 8. A method as claimed inclaim 7, wherein said amino acid is lysine.